Sample Preparation Tips for the Protein Sequencer
Requirements for Protein Sequence Analysis:
The key to obtaining reliable protein sequence information is preparing a sufficient amount of pure material in a form that is compatible with Edman chemistry. Uncertainties in sample recoveries during sample preparation and processing make it difficult to predict the amount of protein required for any given sequencing project. Currently, our instrument can easily sequence at the 5 pmol level and under optimal conditions, identify residues at the 250 fmol level. Ideally, one should consider submitting about 20 to 50 pmol of protein for each sequencing run if possible. It is clear that the methods chosen for sample preparation have a major impact on both the amount of sample required and the overall success of a microsequencing project.
Chemicals that Interfere with Protein Sequence Analysis:
To avoid problems with Edman chemistry, the samples should be free of the following reagents:
1. Buffers and primary amines: Tris buffer is commonly used for protein purification. Tris and glycine are common in samples recovered from SDS-PAGE.
2. Glycerol and sucrose: These reagents are often added to buffers designed for the storage and handling of proteins. These compounds are not volatile and leave a highly viscous residue.
3. Nonionic detergents: Triton X-100, Brij, and Tween solutions often contain aldehydes, oxidants and other contaminates that can inhibit Edman degradation.
4. SDS: Large quantities of SDS can cause instrument malfunction and may lead to the loss of sample from the filter.
Dialysis tubing is often a source of contaminants and other interfering substances. Avoid dialysis as a last step in sample preparation or use thoroughly cleaned, high-quality tubing. Always dialyzed against a salt counterion or dilute acid to prevent the protein and contaminates that may be present from sticking to the tubing.
Sample Loading Conditions:
1. All reagents and solvents must be of the highest purity available (HPLC grade, sequencing grade and electrophoresis grade reagents) to avoid contaminating substances. Avoid molecular biology grade reagents.
2. Always wear gloves and work in a clean dust free area. Dust and finger prints are a major source of contaminating amino acids present in sequencing samples.
3. Avoid drying the sample in glass tubes. This can lead to substantial loss of sample for some proteins. Sample volumes should be less than 150 ul, however with the advent of ABI's Prosorb Sample Preparation Cartridge, sample volumes of up to 750 ul may be sequenced.
4. The sample should be in a volatile solvent or buffer such as acetic acid, formic acid, trifluoroacetic acid, triethylamine, pyridine, acetonitrile, propanol, water, or ammonium bicarbonate (if lyophilized repeatedly).
5. A minimum of 10 to 50 pmol of sample should be analyzed. Our Procise sequencer can sequence 1-2 pmol of sample at its highest level of sensitivity. However, it is more practical to sequence larger amounts of protein to be confident of the sequence obtained or to be confident that the N-terminus is blocked if no sequence is obtained. In most cases the amount of sequenceable material is underestimated by sample loss, inaccurate quantitation, or N-terminal blockage during sample preparation. Therefore, be sure to err on the side of too much sample rather than too little!
6. The sample may contain a small amount of detergent (less than 0.1% SDS). Larger amounts can cause instrument problems.
7. When submitting electroblotted samples on PVDF for sequencing, always try to have as much protein as you can in as small an area of PVDF as possible. Too much PVDF in the sequencer's reaction cartridge can lead to excessive sequencer background.
One reason why the initial yields are often unexpectedly low, is that the amount of sample present is overestimated by the investigator. The most reliable quantitation method is from amino acid analysis. Lowry, BCA, dye binding assays and absorbance are less accurate methods especially in the low microgram amounts.
A Practical Guide to Protein and Peptide Purification. Second Edition. (Paul Matsudaira ed. ), p. 10 - 17, Academic Press, Inc., San Diego, California, 1993.
Additional Protein Sequencing Sample Preparation Tips can be found in A Newcomer's Guide : Preparing Samples for Protein Sequencing at ABI's protein sequencing page.
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